Project Summary/Abstract Exposure to persistent environmental contaminants including polychlorinated biphenyls (PCBs) contributes to metabolic diseases including toxicant-associated steatohepatitis (TASH), a form of nonalcoholic fatty liver disease (NAFLD). PCBs have been positively associated with TASH, hepatocellular cancer, altered liver enzymes, and mortality in human cohorts. A complete understanding of the mechanisms by which PCBs act as a 1st ?hit? in combination with a high fat ?Western diet? (HFD) as a 2nd ?hit? to increase triglyceride accumulation, fibrosis, and inflammation in the liver (hallmarks of TASH) remains elusive. Further, although liver sexual dimorphism is well-established and sex-specific differences in human health outcomes after PCB exposure have been reported, there are few mechanistic studies addressing these differences. N(6)-methyladenosine (m6A) is the most common dynamic modification of transcribed RNAs. The regulation and role of m6A in liver epitranscriptomics and the impact of PCB exposure is unknown. This application addresses RFA-ES-19-002 by investigating the role of altered m6A and its writers, readers, and erasers in TASH in vivo, using an established PCB exposure model. Preliminary RNA-seq data show that liver from HFD + Aroclor 1260 (PCB) exposed male C57BL/6J mice have reduced transcript levels of the m6A methyltransferase complex protein Rbm15 and altered levels of the m6A readers Ythdf1 and Ythdc1/2. Whether these HFD + Aroclor 1260-induced changes are also occur in females and how they affect the m6A epitranscriptome and hepatocyte homeostasis or liver function is unknown. We recently reported increased PCB-induced hepatic inflammation and altered lipid metabolism in female vs. male mice. The goal of this exploratory study is to determine the impact of PCB +/- HFD exposure in vivo on the hepatic m6A epitranscriptome in male and female mice. We will test the hypothesis that altered m6A levels of specific transcripts play a role in TASH in vivo. Integrated analysis will identify m6A epitranscriptome- mediated changes in signaling pathways regulating TASH pathophysiology. This goal will be achieved by 1) Identification of m6A-mediated transcriptome changes in HFD, Aroclor 1260 (a mixture of non-dioxin-like (NDL) PCB subtypes that best mimics the PCB bioaccumulation in human adipose tissue), and HFD + Aroclor 1260 ? exposed male and female mouse liver; 2) Determination if HFD, Aroclor 1260, or HFD + Aroclor 1260 exposure alter the expression of the writers, readers, and erasers of m6A in mouse liver. Integrated analysis will identify m6A epitranscriptome-mediated changes in signaling pathways regulating TASH pathophysiology that may be targets for therapeutic intervention. In the future, knockdown or overexpression of key proteins identified in this study will confirm their role in the observed m6A changes and in downstream signaling.